IL1-beta: a new target for myeloma therapy

ABSTRACT

Diagnostic methods for the detection of multiple myeloma (MM) and the identification of high-risk patients with multiple myeloma-related plasma proliferative disorders, such as MGUS or SMM, likely to progress to active MM are described; The diagnosis is based on the determination of concentrations of bioactive IL-1β produced by the bone marrow plasma cells of these patients. Also described are therapeutic methods for the treatment of MM and for the chemoprevention of the progression from disorders such as MGUS and SMM to active MM, involving the administration of inhibitors of IL-1β.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with Government support from the NationalInstitutes of Health. The Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

The present invention relates generally to the observation of acorrelation between the production of bioactive IL-1β by bone marrowcells and the clinical features of multiple myeloma.

Despite aggressive treatment approaches, multiple myeloma (MM) is auniversally fatal B cell malignancy, accounting for 1-2% of all cancerdeaths. See Silverberg et al. (1996) Ca. J. Clin. 46: 5-27. MM isrecognized clinically by the proliferation of malignant plasma cells inthe bone marrow, the detection of a monoclonal protein (M protein) inthe serum or urine, anemia, hypercalcemia, renal insufficiency and lyticbone lesions. See Kyle and Lust (1989) Seminars in Hematology 26:176-200. Several multiple myeloma-related plasmaproliferative disorders,such as monoclonal gammopathy of undetermined significance (MGUS) andsmoldering multiple myeloma (SMM), are characterized by the detection ofM protein in the serum or urine without the other clinical features ofMM. MGUS, a clinically benign precursor condition of MM is more commonthan MM, occurring in 1% of the population over age 50 and 3% of thoseover age 70. Greipp and Lust (1995) Stem Cells 13: 10-21. It is of greatclinical importance to distinguish patients with MGUS from those withMM, as MGUS patients may be safely observed without resort tochemotherapy. The unnecessary treatment of MGUS patients can lead toacute leukemia or morbidity/mortality from chemotherapy. However, duringlong-term follow-up, of 241 patients with MGUS, 59 patients (24.5%) wenton to develop MM or a related disorder. See Kyle (1993) Mayo ClinicProceedings 68: 28. Thus, the prevention of myeloma from MGUS would havea significant impact on the morbidity and mortality of myeloma.

Chemotherapy and peripheral blood stem cell transplant are thetreatments for symptomatic patients with active MM. Patients who arecandidates for peripheral blood stem cell harvest typically receive fourcycles of VAD (vincristine, doxorubicin [Adriamycin] and dexamethasone)chemotherapy followed by peripheral blood cell harvest. Subsequently,myeloma patients either continue on standard chemotherapy such as VBMCP(vincristine, carmustine [BCNU], melphalan, cyclophosphamide andprednisone) or proceed directly to transplant. See Attal et al. (1996)N. Engl. J. Med. 335: 91 and Alexanian and Dimopoulos (1994) N. Engl. J.Med. 330: 484. Patients who are not candidates for transplantationreceive standard chemotherapy consisting of either VBMCP or melphalan incombination with prednisone. See Oken et al. (1997) Cancer 79: 1561.Recently, thalidomide has been shown to have activity in patients withrecurrent myeloma. See Singhal et al. (2000) N. Engl. J. Med. 2000;342(5): 364 and Singhal et al. (1999) N. Engl. J. Med. 341: 1565.Chemotherapy is usually continued until the patient has reached aplateau state, which is defined as a stable M protein in the serum andurine and no evidence of progression of myeloma. In most patients,chemotherapy plus analgesics can control the bone pain characteristic ofthe disease. The duration of survival of patients with MM ranges from afew months to many years with the median survival of 2.5-3 years. SeeOken et al., supra. Even with transplantation, except for a rarepatient, all patients eventually relapse and succumb to their disease.See Attal et al. supra.

Newer therapeutic strategies for MM are clearly needed. The preventionor delay of the progression to MM from related plasmaproliferativedisorders, such as MGUS and SMM, with an effective chemotherapeuticagent would have a major impact on the treatment of patients withplasmaproliferative disorders.

SUMMARY OF THE INVENTION

The invention is based on the discovery that bioactive IL-1β, whileundetectable in normal plasma cells, is abnormally expressed in theplasma cells of virtually all myeloma patients and approximately 25% ofpatients with related plasma proliferative disorders, such as MGUS orSMM. The invention includes diagnostic methods for the detection of MMand the identification of high-risk MGUS and SMM patients likely toprogress to active MM. The diagnosis is based on the determination ofconcentrations of bioactive IL-1β produced by the bone marrow plasmacells of these patients. The invention also includes therapeutic methodsfor the treatment of myeloma and for the chemoprevention of theprogression from multiple myeloma-related plasmaproliferative disorders,such as MGUS or SMM, to active MM, involving the administration ofinhibitors of IL-1β.

In general, the invention features a method of quantitating IL-1β in abone marrow preparation including culturing stromal cells with the bonemarrow preparation, determining the amount of IL-6 produced by thestromal cell culture and correlating the amount of IL-6 produced to theIL-1β concentration in the bone marrow preparation by comparison to astandard curve prepared by measuring IL-6 produced by stromal cellscontacted with known concentrations of IL-1β. The bone marrowpreparation can include, but is not limited to, a fresh supernatant fromcultured bone marrow cells, a previously frozen supernatant fromcultured bone marrow cells and a mononuclear cell preparation purifiedfrom bone marrow. The bone marrow preparation can be from, but is notlimited to, a patient suffering from a MM or a multiple myeloma-relatedplasmaproliferative disorder.

In another embodiment, the invention features a method of detecting MMin an individual including culturing stromal cells with a bone marrowpreparation from the individual, determining the amount of IL-6 producedby the stromal cell culture, wherein an elevated level of IL-6 isindicative of MM. The invention also features a method of identifying apatient with a multiple myeloma-related plasmaproliferative disorderlikely to progress to active MM including the steps of culturing stromalcells with a bone marrow preparation from the patient, determining theamount of IL-6 produced by the stromal cell culture, wherein an elevatedlevel of IL-6 is indicative of a likelihood the patient will progress toactive MM. The multiple myeloma-related plasmaproliferative disorder canbe, but is not limited to, MGUS or SMM. An elevated level of IL-6 canbe, but is not limited to, a concentration of IL-6 greater than thatproduced by stromal cells incubated with 1 pg/ml of recombinant IL-1β.The bone marrow preparation can be, but is not limited to, a freshsupernatant from cultured bone marrow cells, a previously frozensupernatant from cultured bone marrow cells and a mononuclear cellpreparation purified from bone marrow. An inhibitor of IL-1β can beadded to the step of culturing the stromal cell culture with the bonemarrow preparation. This inhibitor of IL-1β can be, but is not limitedto, an anti-ILβ antibody, a soluble IL-1 receptor (sIL-1R) type I, asIL-1R type II, an interleukin-1 receptor antagonist (IL-1ra) and anIL-1 TRAP.

The invention also features a method of monitoring the effectiveness ofthe treatment of a patient with a MM or a multiple myeloma-relatedplasmaproliferative disorder including culturing stromal cells with abone marrow preparation from the patient after the initiation oftreatment, determining the amount of IL-6 produced by the stromal cellculture and comparing the amount of IL-6 with a known standard or apatient determined standard.

In another embodiment, the invention features a method of identifying apatient with a multiple myeloma-related plasmaproliferative disorderlikely to progress to active MM including culturing a bone marrowpreparation from the patient with a T-cell line which produces IL-2 inresponse to IL-1β, determining the amount of IL-2 produced by the T-cellline, wherein an elevated level of IL-2 is indicative of a likelihoodthe patient will progress to active MM. The multiple myeloma-relatedplasmaproliferative disorder can be, but is not limited to, MGUS or SMM.The T-cell line can be, but is not limited to, EL4.6.1, LBRM 33 andprimary cultures of thymocytes.

In another aspect, the invention features methods of treating patientsby administering an inhibitor of IL-1β. This includes, but is notlimited to, the treatment of patients with MM, MGUS, SMM and indolentmultiple myeloma (IMM). It also includes administering an inhibitorIL-1β to patients in amounts effective to inhibit the production of IL-6by patient bone marrow stromal cells or to inhibit myeloma cellproliferation. In another embodiment, the invention features methods ofinhibiting the progression from MGUS or SMM to active MM byadministering an inhibitor of IL-1β. The inhibitor of IL-1β used inthese treatment methods can include, but is not limited to, an anti-ILβantibody, a soluble receptor (sIL-1R) type I, a sIL-1R type II, aninterleukin-1 receptor antagonist (IL-1 ra) or an IL-1 TRAP.

In another aspect, the invention features kits including an inhibitor ofbioactive IL-1β, a negative control for the inhibitor of bioactive IL-1βand a positive control for bioactive IL-1β. In these kits the inhibitorof bioactive IL-1β can be, but is not limited to, an anti-ILβ antibody,a soluble IL-1 receptor (sIL-1R) type I, a sIL-1R type II, aninterleukin-1 receptor antagonist (IL-1ra) or an IL-1 TRAP. The positivecontrol for bioactive IL-1β can be, but is not limited to, recombinantIL-1β. The kits can include bone marrow stromal cells. The kits can alsoinclude a label or package insert indicating that the positive controlfor bioactive IL-1β can be used to prepare a standard curve of IL-6produced by stromal cells contacted with known concentrations ofbioactive IL-1β.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice and testing of the present invention, suitable methods andmaterials are described. All publications, patent applications, patentsand other references mentioned herein are incorporated by reference intheir entirety. In case of conflict, the present specification,including definitions, will control. In addition, the materials,methods, and examples are illustrative only and are not intended to belimiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic depicting the role of IL-1β expression in theprogression of MGUS to MM.

FIG. 2 is a schematic representation of the bioassay for IL-1β inducedIL-6 production.

FIG. 3 shows levels of IL-1β induced IL-6 production in MGUS, SMM and MMpatients. An anti-IL-1β antibody is used as an inhibitor of IL-1βbioactivity.

FIG. 4 shows levels of IL-1β induced IL-6 production in MGUS and MMpatients. Both an anti-IL-1β antibody and IL-1 TRAP are used asinhibitors of IL-1β bioactivity.

FIGS. 5A and 5B show levels of IL-1β induced IL-6 production in MGUS,SMM and MM patients. Both an anti-IL-1β antibody and IL-1 TRAP are usedas inhibitors of IL-1β bioactivity. VEGFTRAP is used as a negativecontrol.

FIG. 6 shows the levels of IL-6 produced by stromal cells co-culturedwith normal, MGUS, SMM and MM patient supernatants.

DETAILED DESCRIPTION

The current invention is based on the observation that the biologicaleffects of IL-1β closely parallel the clinical features of humanmyeloma. Interleukin-6 (IL-6) has been shown to be the central growthfactor for myeloma cells and is produced by bone marrow stromal cells;See Kawano et al., (1988) Nature 332: 83; Schwab et al. (1991) Blood 77:587; Klein et al. (1989) Blood 73: 517; and Portier et al. (1991) Eur.J. Immunol. 21: 1759. Although many cytokines can stimulate IL-6production, IL-1β appears to be the major cytokine responsible for theparacrine production of IL-6 by marrow stromal cells in myeloma. SeeCarter et al. (1990) Br. J. Haematol. 74: 424. IL-1β is expressed by theplasma cells of virtually all myeloma patients; however, it is notproduced by normal plasma cells. See Lust and Donovan (1999)Hematology-Oncology Clinics of North America 13: 1117; Donovan et al.(1998) Leukemia 12: 593; and Lacy et al. (1999) Blood 93: 300.

The current invention is based on the observation that aberrant IL-1βproduced by myeloma cells is able to induce IL-6 production by bonemarrow stromal cells and the IL-6 stimulates myeloma cells to grow. Thisparacrine model of IL-6 production suggests a therapeutic approach forthe treatment of myeloma: inhibition of IL-1β induced IL-6 productionwith an IL-1β inhibitor. Such treatment with an IL-1 inhibitor willreduce plasma cell growth and slow or reverse further progression of thedisease.

As shown in Table 1, specific criteria exist that serve as usefulguidelines to allow clinicians to differentiate between MM and othermultiple myeloma-related plasma proliferative disorders. Multiplemyeloma-related plasmaproliferative disorders are defined to includeMGUS, smoldering MM (SMM) and indolent MM (IMM). Patients with MGUS, aclinically benign condition, usually have less than 10% marrow plasmacells, a serum monoclonal protein <3 gm/dL, no urinary Bence Jonesprotein and no anemia, renal failure, lytic bone lesions, orhypercalcemia. In contrast, patients with active MM present with amarrow plasmacytosis of ≧10%, a serum monoclonal protein of ≧3 gm/dL, a24-hour urine monoclonal protein of ≧1 gm, and lytic bone lesions. MMpatients also often present with back pain, severe fatigue, pneumonia,or bone pain. Kyle and Lust (1989) Seminars in Hematology 26: 176.Patients with SMM are usually asymptomatic. They have a marrowplasmacytosis of ≧10% and/or a serum monoclonal protein of ≧3 gm/dL.Lytic bone lesions are absent and they have stable disease. See Kyle andGreipp (1980) N. Engl. J. Med. 302: 1347. Patients with IMM are similarto those with SMM, except bone lesions may be present on bone surveystudies. In contrast to MM patients, who all receive chemotherapy,patients with MGUS, SMM and IMM have stable disease and are followed offchemotherapy. However, patients who have a serum monoclonal proteinof >2.0 g/dL are at high risk of eventually developing active MM,requiring chemotherapy. Such patients are candidates for noveltherapeutic strategies to inhibit or prevent the development of activeMM. TABLE 1 Characteristic MM SMM MGUS Marrow plasma cells ≧10% ≧10%<10% Serum M-spike ≧3 g/dL ≧3 g/dL <3 g/dL Bence Jones Protein ≧1 g/24 h<1 g/24 h <1 g/24 h Anemia Usually Present May be Present AbsentHypercalcemia, Renal May be Present Absent Absent Insufficiency Lyticbone lesions Usually Present Absent Absent

During long-term follow-up of 241 patients with MGUS, 59 patients(24.5%) went on to develop MM or a related disorder. See Kyle (1993)Mayo Clinic Proc. 68: 26-36. Closer examination of the individuals thatprogressed to MM revealed that the majority of these patients remainedstable for an extended period of time and then subsequently progressedto overt MM over a relatively short period of time. Of the 59 patientsthat progressed, 39 went on to develop MM. Of these 39 patients, 18 hadserial serum studies performed. In these eighteen patients, theM-protein remained stable for a median of 8 years and then increasedslowly over 1-4 years in 11 patients or rapidly in less than 1 year inseven patients.

As summarized in Table 2, supernatants of bone marrow cells from normalindividuals stimulated the production of minimal amounts of IL-6 bymarrow stromal cells. Supernatants from MGUS patients generated low, butdetectable, amounts of IL-6, slightly above normal individuals. Allmyeloma patients studied produced IL-6 levels above normal individualsand some were 100-200 fold elevated. The SMM patients appeared to fallinto two groups: those with high IL-6 values (4 patients) and those withlow values (10 patients). Two of the four patients with high values ofIl-1 β induced IL-6 production have subsequently progressed to activemyeloma within two years. Thus, up regulation of IL-1β productionappears critical in the progression from MGUS or SMM to active MM. SeeFIG. 1. TABLE 2 IL-6 Production From Stromal Cell Co-cultures DiagnosisNumber Average IL-6 (pg/ml) IL-6 Range (pg/ml) Normal 2 114 110-117 MGUS 5 426 114-1258 SMM 10 1711 154-3474 SMM 4 23198 6987-35000 MM 1010775  610-27297

The invention features an assay for bioactive IL-1β that allows for thequantitative detection of functional, bioactive IL-1β in supernatants ofbone marrow cells taken from patients with plasmaproliferativedisorders. As shown in FIGS. 3-6, studies on bone marrow cells frompatients with MGUS, SMM and active MM indicate that this assay will beuseful as a biomarker in differentiating those patients with stableMGUS/SMM from those progressing to active MM.

IL-1β

Interleukin-1 (IL-1) is produced predominantly by peripheral bloodmonocytes during an inflammatory response and exists in two distinctforms, IL-1α and IL-1β. For a review, see Dinarello (1996) Blood 87:2095. IL-1β is synthesized as a biologically inactive precursor,proIL-1β, which lacks a conventional leader sequence and is notprocessed by a signal peptidase. See March (1985) Nature 315: 641.proIL-1β is cleaved by an intracellular cysteine protease betweenAsp-116 and Ala-117 to produce the biologically active C-terminalfragment found in human serum and synovial fluid. See Sleath et al.(1992) J. Biol. Chem. 265: 14526 and Howard et al. (1991) J. Immunol.147: 2964. Bioactive IL-1β includes, but is not limited to, thebiologically active C-terminal fragment of IL-1β after cleavage betweenAsp-116 and Ala-117.

Assays for Bioactive IL-1β

This invention provides methods of detecting patients with MM and foridentifying patients likely to transform from multiple myeloma-relatedplasmaproliferative disorders, such as MGUS or SMM, to active MM. Alsoincluded are methods of monitoring the effectiveness of the treatment ofa patient with MM or a multiple myeloma-related plasmaproliferativedisorder comprising quantitating levels of bioactive IL-1β produced bypatient bone marrow plasma cells at various time points in treatment.These time points include, but are not limited to, prior to the start oftreatment and anytime point after treatment has commenced. Such methodsinvolve the quantitation of bioactive IL-1β produced by bone marrowplasma cells. Any art known assay for the quantitation of bioactiveIL-1β is included within the scope of the invention. Such assaysinclude, but are not limited to, osteoclast activation assays, T-cellassays and stromal cell assays.

T-Cell Bioassay

As outlined in Cytokines: A Practical Approach, ed. F. R. Blackwill,Oxford University Press (1991) pp. 311-12, bioassays for functional IL-1can utilize the ability of IL-1β to induce IL-2 production by T-celllines, such as EL4.6.1 and LBRM 33, or by primary cultures of thymocytes(LAF assay). Alternatively, IL-1 can be assayed by measuring its abilityto stimulate the proliferation of lines such as the subclone D10S of themurine T-helper cell-line D10.64.1. A subclone, NOB-1, of the murinethymoma cell-line EL4.6.1 provides a simple and sensitive bioassay forIL-1. This line produces IL-2 in response to IL-1, which is measuredusing the CTLL-2 assay. See Cytokines: A Practical Approach, ed. F. R.Blackwill, Oxford University Press (1991) pp. 311-312.

Stromal Cell Bioassay for IL-6

The stromal cell bioassay for IL-1β quantitates the amount of IL-6produced by stromal cells cultured with bone marrow preparations frompatients with various plasmaproliferative disorders. The assay isdiagramed in FIG. 2. The assay comprises culturing bone marrow stromalcells with patient bone marrow preparations and determining the amountof IL-6 produced by the stromal cells. The amount of IL-6 produced isthen correlated to the concentration of IL-1β in the bone marrowpreparation by comparison to a standard curve prepared by measuring IL-6produced by stromal cells contacted with known concentrations of IL-1β.

Normal bone marrow stromal cells can be obtained commercially, forexample, from Clonetics, Walkersville, Md. Bone marrow preparationsinclude, but are not limited to, fresh supernatants from patient bonemarrow cells, previously frozen supernatants from patient bone marrowcells and fresh mononuclear cell preparations of patient bone marrowcells. It can be technically difficult to have marrow stromal cellsconstantly available when patient bone marrow samples are drawn. Thus,it is often convenient to culture bone marrow cells, collect thesupernatants and store the supernatants at −80° C. until stromal cellsare available. However, when stromal cell cultures are available,patient bone marrow cells can be plated directly onto stromal cells.Stromal cells are cultured with either IL-1β standards or patientsupernatants, with or without the addition of an anti-IL-1β antibody orother IL-1β inhibitor. Recombinant IL-1β, obtained commercially, forexample, from R&D Systems, Inc., Minneapolis, Minn., at concentrationsincluding, but not limited to, 0.1 pg/ml, 1.0 pg/ml, 10 pg/ml, 100 pg/mland 1000 pg/ml can be used to prepare a standard curve of IL-6 producedby stromal cells contacted with known concentrations of IL-1β.

Stromal cell supernatants are analyzed for IL-6 concentrations using anyart known assay for the quantitation of IL-6. One such assay is thehuman IL-6 ELISA kit from BioSource International, Inc., Camarillo,Calif. (Catalog Numbers KHC0061, KHC0062, KHC0063 or KHC0064). All suchassays for the quantitation of IL-6 are performed according to themanufacturer's specifications. An elevated level of IL-6 can be, forexample, a concentration of IL-6 greater than that produced by stromalcells incubated with a standard concentration of recombinant IL-1β, suchas, for example, 0.1 pg/ml, 1 pg/ml or 10 pg/ml recombinant IL-1β. Anelevated level of IL-6 can also be, for example, a concentration of IL-6greater than that produced by stromal cells from normal, healthyindividuals. Alternatively, an elevated level of IL-6 can also bedetermined by a patient specific standard; for example, an IL-6concentration is elevated if it is greater than the IL-6 concentrationproduced by stromal cells obtained from the same patient at an earlieror later point in time.

Therapeutic Methods

This invention provides methods for the treatment of patients with MMcomprising the administration of inhibitors of IL-1β. The invention alsoprovides methods of chemoprevention, i.e., preventing the transitionfrom MGUS or SMM to MM comprising the administration of inhibitors ofIL-1β.

Inhibitors of IL-1β

Inhibitors of bioactive IL-1β include, but are not limited to,antibodies that bind to IL-1β, antibodies that bind to the IL-1 receptoror to peptide fragments of the receptor, the IL-1 receptor antagonistprotein (IL-1ra), soluble IL-1 receptor (sIL-1R), peptide fragments ofthe IL-1 receptor, and chimeric molecules such as IL-1 TRAP.

Antibodies to IL-1β

Antibodies having specific binding affinity for IL-1β can be producedthrough standard methods. Alternatively, antibodies may be commerciallyavailable, for example, from R&D Systems, Inc., Minneapolis, Minn. Theterms “antibody” and “antibodies” include polyclonal antibodies,monoclonal antibodies, humanized or chimeric antibodies, single chain Fvantibody fragments, Fab fragments, and F(ab)₂ fragments. Polyclonalantibodies are heterogeneous populations of antibody molecules that arespecific for a particular antigen, which are contained in the sera ofthe immunized animals. Polyclonal antibodies are produced usingwell-known methods.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular epitope contained within an antigen, can be preparedusing standard hybridoma technology. In particular, monoclonalantibodies can be obtained by any technique that provides for theproduction of antibody molecules by continuous cell lines in culturesuch as described by Kohler, G. et al., Nature, 1975, 256: 495, thehuman B-cell hybridoma technique (Kosbor et al., Immunology Today, 1983,4: 72; Cole et al., Proc. Natl. Acad. Sci. USA, 1983, 80: 2026), and theEBV-hybridoma technique (Cole et al., “Monoclonal Antibodies and CancerTherapy”, Alan R. Liss, Inc., 1983, pp. 77-96). Such antibodies can beof any immunoglobulin class including IgG, IgM, IgE, IgA, IgD, and anysubclass thereof. The hybridoma producing the monoclonal antibodies ofthe invention can be cultivated in vitro or in vivo.

A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine monoclonal antibody and a humanimmunoglobulin constant region. Chimeric antibodies can be producedthrough standard techniques.

Antibody fragments that have specific binding affinity for IL-1β can begenerated by known techniques. For example, such fragments include, butare not limited to, F(ab′)₂ fragments that can be produced by pepsindigestion of the antibody molecule, and Fab fragments that can begenerated by reducing the disulfide bridges of F(ab′)₂ fragments.Alternatively, Fab expression libraries can be constructed. See, forexample, Huse et al., 1989, Science, 246: 1275. Single chain Fv antibodyfragments are formed by linking the heavy and light chain fragments ofthe Fv region via an amino acid bridge (e.g., 15 to 18 amino acids),resulting in a single chain polypeptide. Single chain Fv antibodyfragments can be produced through standard techniques. See, for example,U.S. Pat. No. 4,946,778.

IL-1ra

The IL-1 receptor antagonist (IL-1 ra) (also called IRAP, for IL-1receptor antagonist protein) is a third member of the IL-1 family. Itacts as a natural antagonist of IL-1α and IL-1β by binding to the IL-1receptor but not transducing an intracellular signal or a biologicalresponse. The gene encoding this antagonist of IL-1 has been described.See Hannum et al. (1990) Nature 343: 336-340; Eisenberg et al. (1990)Nature 343: 341-346; and Carter et al. (1990) Nature 344: 633-638.IL-1ra inhibits the biological activities of IL-1 both in vitro and invivo, and has been shown to be effective in animal models of septicshock, rheumatoid arthritis, graft versus host disease, stroke, cardiacischemia; it is currently in clinical trials for some of theseindications. For a review, see Dinarello and Thompson (1991) Immunol.Today 12: 404-410. In vivo administration of IL-1ra is well tolerated.Normal animals, including humans, can be infused intravenously with highdoses of this protein without any change in physiological or metabolicparameters. For example, human volunteers infused with 133 mg/h IL-1rafor 72 hours exhibited no change in clinical or laboratory values. SeeDinarello et al. (1993) J. Amer. Med. Assoc. 269: 1829-1835.

sIL-1R

There are three known IL-1 receptor subunits. The active receptorcomplex consists of the type I receptor and IL1RAcP (for IL-1 receptoraccessory protein). The type I receptor is responsible for binding ofthe three naturally occurring ligands (IL-1α, IL-1β and IL-1ra) and isable to do so in the absence of the IL1RAcP. However, signaltransduction requires interaction of IL-1α or IL-1β with the IL1RAcP.IL-1ra does not interact with the IL-1RAcP and hence cannot signal. Athird receptor subunit, the type II receptor, binds IL-1α or IL-1β, butcannot signal due to its lack of an intracellular domain. Rather it actsas a decoy either in its membrane form or as an antagonist in a cleavedsecreted form, thus inhibiting IL-1 activity. For a review, seeDinarello (1996) Blood, 87: 2095-2147.

Also included as inhibitors of bioactive IL-1β are peptidescorresponding to portions of the IL-1 receptor. These include, but arenot limited to, synthetic peptides corresponding to residues 86-93 ofthe human type I IL-1 receptor, which bind IL-1 (α and β) and inhibitIL-1 activity in vitro and in vivo. See Tanihara et al. (1992) Biochem.Biophys. Res. Commun. 188: 912.

IL-1 TRAP

The IL-1 TRAP is as essentially described in U.S. Pat. No. 5,844,099.Briefly, the IL-1 TRAP is a fusion protein comprising the human cytokinereceptor extracellular domains and the Fc portion of human IgG1. TheIL-1 TRAP incorporates into a single molecule the extracellular domainsof both receptor components required for IL-1 signaling; the IL-1 Type Ireceptor (IL-1RI) and the IL-1 receptor accessory protein (AcP). Sinceit contains both receptor components, the IL-1 TRAP binds IL-1β andIL-1α with picomolar affinity, while the IL-1RI alone in the absence ofAcP binds with ˜1 nM affinity. The IL-1 TRAP was created by fusing thesequences encoding the extracellular domains of the AcP, IL-1RI, and Fcinline without any intervening linker sequences. An expression constructencoding the fusion protein is transfected into Chinese hamster ovary(CHO) cells, and high producing lines are isolated that secrete the IL-1TRAP into the medium. The IL-1 TRAP is a dimeric glycoprotein with aprotein molecular weight of 201 kDa and including glycosylation has atotal molecular weight of ˜252 kDa. The dimer is covalently linked bydisulfide bonds in the Fc region.

Rheumatoid arthritis is a chronic autoimmune disease in which the immunesystem attacks the tissue that lines and cushions the joints. ExcessIL-1β production mediates the cartilage and joint damage associated withrheumatoid arthritis. In preclinical trials, the IL-1 TRAP was a potentblocker of IL-1 activity, has a long half-life in the blood, penetratesinto the joints and blocks IL-1-induced cartilage erosion in animalmodels. A placebo controlled, double blind, dose-escalation Phase Itrial is currently underway to assess the safety and tolerability of theIL-1 TRAP for the treatment of rheumatoid arthritis. Seehttp://regeneron.com.

Pharmaceutical Compositions

Effective doses of an IL-1β inhibitor useful for treating MM andmultiple myeloma-related plasmaproliferative disorders may be determinedusing methods well known in the art. See for example Fingle et al., ThePharmaceutical Basis of Therapeutics, Goodman and Gilman, eds. MacMillanPublishing Co., New York, pp. 1-46 (1975) or “Remington's PharmaceuticalScience.” Pharmaceutical compositions for use according to the currentinvention include the IL-1β inhibitor in a pharmaceutically acceptableliquid (including, but not limited to sterile water, phosphate bufferedsaline or dextrose solution), solid (e.g. wax) or semi-solid carrier(e.g. gelfoam), incorporated into liposomes, microcapsules or controlledrelease preparations (including inhibitor expressing cells). Theadministration route may be any mode of administration known in the art,including, but not limited to, intravenously, intrathecally,subcutaneously, by injection into involved tissue, intraarterially,intranasally, orally, or via an implanted device. Pharmaceuticalcompositions of this invention comprise any of the compounds of thepresent invention, and pharmaceutically acceptable salts thereof, withany pharmaceutically acceptable carrier, adjuvant or vehicle.

A therapeutically effective amount is an amount capable of producing amedically desirable result in a treated mammal, e.g., a human patient.The dosage range required depends on the individual IL-1β inhibitorselected, the route of administration, the nature of the formulation,the nature of the subject's condition, and the judgment of the attendingpractitioner. Wide variations in the needed dosage are to be expected inview of the variety of compounds available and the differingefficiencies of various routes of administration. For example, oraladministration would be expected to require higher dosages thanadministration by intravenous injection. Dosages will vary, butpreferred dosage for administration is from approximately 0.01 to 1,000mg/kg of body weight. For example, a preferred dosage can be, but is notlimited to, 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, 10, 25, 50, 100, 500, 750 or1,000 mg/kg of body weight. Variations in these dosage levels can beadjusted using standard empirical routines for optimization, as is wellunderstood in the art. Specific dosage and treatment regimens for anyparticular patient will depend upon a variety of factors, including theactivity of the specific compound employed, the age, body weight,general health status, sex, diet, time of administration, rate ofexcretion, drug combination, the severity and course of the disease, andthe patient's disposition to the disease and the judgment of thetreating physician.

Typically, the pharmaceutical compositions of this invention will beadministered from about 1 to 5 times per day or, alternatively, as acontinuous infusion. Such administration can be used as a chronic oracute therapy. The amount of active ingredient that may be combined withthe carrier materials to produce a single dosage form will varydepending upon the host treated and the particular-mode ofadministration. A typical preparation will contain from about 5% toabout 95% active compound (w/w). Preferably, such preparations containfrom about 20% to about 80% active compound.

The IL-1β inhibitors of the present invention may be employed alone orin conjunction with other therapeutic agents. Such therapeutic agentsmay include, but are not limited to, additional IL-1β inhibitors,chemotherapy agents and analgesics. Such agents may be administeredprior to, sequential to or concurrent with the administration of theIL-1β inhibitor to the patient.

Kits and Articles of Manufacture

In further embodiments, the present invention includes kits or articlesof manufacture for conveniently and effectively carrying out the methodsin accordance with the present invention.

Inhibitors of IL-1β can be packaged as kits or articles of manufacturethat contain one or more unit dosage forms. Each kit or article ofmanufacture typically comprises at least one container with a label.This container may, for example, comprise glass or plastic bottles orvials or metal or plastic foil. Another example of such a kit is a“blister pack.” Blister packs are well known in the packaging industryand are widely used for packaging pharmaceutical unit dosage forms. Thepackage may also be accompanied by a label or packaging insert givinginstructions for the intended use of the enclosed IL-1β inhibitor, forexample, indicating that the inhibitor may be administered for thetreatment of plasmaproliferative disorders, such as MM, MGUS or SMM.These directions may indicate that the IL-1β inhibitor may be used aloneor in combination with other agents. The package may also include theseother agents. The package may also include materials desirable from acommercial or user standpoint, including buffers, filters, needles,syringes.

The scope of the invention also includes kits containing materialsnecessary for the determination of the concentration of IL-1β present inbone marrow preparations and other supernatants. These kits comprise apositive control for bioactive IL-1β and an inhibitor of bioactiveIL-1β. The kits may further comprise a negative control for theinhibitor of bioactive IL-1β. This negative control may be, but is notlimited to, VEGF TRAP. The VEGF TRAP is a fusion protein comprising thereceptor for vascular endothelial growth factor (VEFGF) and portions ofhuman IgG, prepared as essentially described in U.S. Pat. No. 5,844,099.See also, http://regeneron.com. The kits may further comprise bonemarrow stromal cells. The positive control for bioactive IL-1β may be,but is not limited to, recombinant IL-1β. The inhibitor of bioactiveIL-1β may be, but is not limited to an anti-IL-1β antibody, a solubleIL-1 receptor (sIL-1R) type I, a sIL-1R type II, an interleukin-1receptor antagonist (IL-1ra) or IL-1 TRAP. The kit may include a labelor packaging insert indicating that the positive control for bioactiveIL-1β may be used to prepare a standard curve of IL-6 produced bystromal cells contacted with known concentrations of bioactive IL-1β.

EXAMPLES Example 1 IL-6 Produced by Stromal Cells Cultured with PatientBone Marrow Cells with the Addition of an Anti-IL-1β Antibody as anInhibitor of Bioactive IL-1β

Ficoll purified bone marrow cells from patients with variousplasmaproliferative disorders were cultured at 2×10⁶ cells/ml for 48hours. Supernatants were frozen at −80° C. until assayed on stromalcells. Normal stromal cells (Clontech, Walkersville, Md.) were plated at1×10⁵ cells/ml and incubated at 37° C. for 48 hours. After 48 hours, thestromal cells were washed and incubated with patient supernatants, withor without a polyclonal neutralizing anti-IL-1β antibody. Cultures wereincubated at 37° C. for an additional 48 hours. Supernatants wereharvested and frozen at −20° C. until analysis of IL-6 production.Supernatants were analyzed for IL-6 using a human IL-6 ELISA kit(BioSource International, Inc., Camarillo, Calif.) according to themanufacturer's specifications. Results are shown in FIG. 3.

Example 2 IL-6 Produced by Stromal Cells Cultured with Patient BoneMarrow Cells with the Addition of an IL-1 TRAP as an Inhibitor ofBioactive IL-ID

Ficoll purified bone marrow cells from patients with variousplasmaproliferative disorders were cultured at 2×10⁶ cells/ml for 48hours. Supernatants were frozen at −80° C. until assayed on stromalcells. Normal stromal cells (Clontech, Walkersville, Md.) were plated at1×10⁵ cells/ml and incubated at 37° C. for 48 hours. After 48 hours, thestromal cells were washed and incubated with either IL-1β standards orpatient supernatants, with or without IL-1 TRAP as an inhibitor ofbioactive IL-1β. Cultures were incubated at 37° C. for an additional 48hours. Supernatants were harvested and frozen at −20° C. until analysisof IL-6 production. Supernatants were analyzed for IL-6 using a humanIl-6 ELISA kit (BioSource International; Inc., Camarillo, Calif.)according to the manufacturer's specifications. To prepare a standardcurve of IL-6 produced by known concentrations of IL-1β, bone marrowstromal cells were incubated with media or recombinant IL-1β in thefollowing concentrations; 1000, 100, 10, 1.0 or 0.1 pg/ml. Results areshown in FIG. 4.

Example 3 IL-6 Produced by Stromal Cells Cultured with Patient BoneMarrow Cells with the Addition of an Anti-IL-11 Antibody, an IL-1 TRAPor a VEGF TRAP

IL-6 produced by stromal cells cultured with bone marrow cells frompatients with various plasma proliferative diseases with the addition ofan anti-IL-1β polyclonal antibody, an IL-1 TRAP or a VEGF TRAP are shownin FIGS. 5A and 5B. The assay was performed as outlined in Examples 1and 2, above. Patient supernatants were assayed in a total volume of 0.5ml as follows:

-   -   0.5 ml patient supernatant+media alone    -   0.5 ml patient supernatant+10 micrograms/well anti-IL-1 antibody    -   0.5 ml patient supernatant+1 microgram/well IL-1 TRAP    -   0.5 ml patient supernatant+1 microgram/well VEGF TRAP

The supernatants were harvested and an IL-6 ELISA was performed. Asshown in FIG. 5B, supernatants from normal patients or those with MGUSinduce only small amounts of IL-6. In contrast, as shown in FIGS. 5 aand 5B, patients with MM induce IL-6 levels equivalent to 1-10 pg ofIL-1β. Some SMM patients may generate IL-6 levels similar to normal/MGUSpatients while others may induce IL-6 levels similar to patients withMM. SMM patients with high levels of IL-6 induction have been observedto progress to active MM in 1-2 yrs requiring chemotherapy. The IL-1TRAP was superior to the anti-IL-1β antibody at inhibiting IL-6production by patient supernatants especially in view of the fact that10 fold more antibody was utilized. The IL-1 TRAP was very effective atinhibiting IL-6 production in all patients with MM or SMM that hadelevated IL-6 levels. For example, the first MM patient shown in FIG. 5Aproduced 131,512 pg/ml of IL-6 that was inhibited to 14,873 pg/ml by theaddition of IL-1 TRAP, a reduction of 93% compared to the media control.The VEGF TRAP caused no reduction in IL-6 production and may haveslightly stimulated IL-6 production in certain cases. The first SMMpatient shown on FIG. 5A indicate an IL-6 level of 82,084 pg/ml, whichwas reduced to 15,672 pg/ml with the addition of an IL-1 TRAP (a 88%reduction). This individual is likely to progress to active MM based onthe high IL-6 level generated by the patient supernatant. In vitroresults demonstrating a significant reduction in IL-6 levels with theaddition of the IL-1 TRAP indicate that this patient may benefit fromtherapy with a potent IL-1 inhibitor, such as the IL-1 TRAP.

Example 4 IL-6 Produced by Stromal Cells Cultured with Bone Marrow Cellsfrom a Variety of Patients

FIG. 6 is a summary of the level of IL-6 produced by bone marrow cellsfrom a collection of patients with MM, MGUS and SMM. The same assaysystem outlined in Examples 1 and 2 was used. Supernatants of bonemarrow cells from normal individuals stimulated the production ofminimal amounts of IL-6 by marrow stromal cells. Therefore, purificationof myeloma cells from patient marrows was not necessary. Supernatantsfrom MGUS patients generated low but detectable amounts of IL-6 slightlyabove normal individuals. All MM patients studied produced IL-6 levelsabove normal individuals and some were 100-200 fold elevated. Allpatients with MM had bone lesions evident on X-ray. Interestingly, oneMM patient with an IL-6 value of 25,962 had a labeling index of 1.4% anda second MM patient with an IL-6 value of 27,297 had a labeling index of5%. The SMM patients appeared to fall into two groups: those with highvalues of IL-6 production (4 patients) and those with low values of IL-6production (10 patients). Two of the four patients with high values ofIL-6 production have subsequently progressed to active myeloma withintwo years. These results indicate that IL-1β induced IL-6 production isa useful criteria for differentiating those patients that will progressfrom MGUS/SMM to active MM.

Other Embodiments

It is to be Understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1-16. (canceled)
 17. A method of inhibiting interleukin-6 (IL-6)production by bone marrow stromal cells in an individual diagnosed withmultiple myeloma or a multiple myeloma-related plasmaproliferativedisorder, said method comprising administering an inhibitor ofinterleukin-1β to said individual in an amount effective to inhibit theproduction of IL-6 by said bone marrow stromal cells.
 18. A method ofinhibiting interleukin-6 induced myeloma cell proliferation in anindividual diagnosed with multiple myeloma or a multiple myeloma-relatedplasmaproliferative disorder, said method comprising administering aninhibitor of interleukin-1β to said individual in an amount sufficientto inhibit myeloma cell proliferation.
 19. The method of claim 17,wherein said multiple myeloma-related plasmaproliferative disorder isselected from the group consisting of monoclonal gammopathy ofundetermined significance, smoldering multiple myeloma and indolentmultiple myeloma.
 20. A method of inhibiting the progression from amultiple myeloma-related plasmaproliferative disorder to multiplemyeloma in an individual diagnosed with a multiple myeloma-relatedplasmaproliferative disorders said method comprising administering aninhibitor of interleukin-1β to said individual.
 21. (canceled)
 22. Themethod of claim 17, wherein said inhibitor of IL-1β is selected from thegroup consisting of an anti-ILβ antibody, a soluble IL-1 receptor(sIL-1R) type I, a sIL-1R type II, an interleukin-1 receptor antagonistand an IL-1 TRAP. 23-27. (canceled)
 28. The method of claim 18, whereinsaid multiple myeloma-related plasmaproliferative disorder is selectedfrom the group consisting of monoclonal gammopathy of undeterminedsignificance, smoldering multiple myeloma and indolent multiple myeloma.29. The method of claim 20, wherein said multiple myeloma-relatedplasmaproliferative disorder is monoclonal gammopathy of undeterminedsignificance.
 30. The method of claim 20, wherein said multiplemyeloma-related plasmaproliferative disorder is smoldering multiplemyeloma.
 31. The method of claim 17, wherein said inhibitor of IL-1β isan interleukin-1 receptor antagonist.
 32. The method of claim 18,wherein said inhibitor of IL-1β is selected from the group consisting ofan anti-ILβ antibody, a sIL-1R type I, a sIL-1R type II, aninterleukin-1 receptor antagonist, and an IL-1 TRAP.
 33. The method ofclaim 18, wherein said inhibitor of IL-1β is an interleukin-1 receptorantagonist.
 34. The method of claim 19, wherein said inhibitor of IL-1βis selected from the group consisting of an anti-ILβ antibody, a sIL-1Rtype I, a sIL-1R type II, an interleukin-1 receptor antagonist, and anIL-1 TRAP.
 35. The method of claim 19, wherein said inhibitor of IL-1βis an interleukin-1 receptor antagonist.
 36. The method of claim 20,wherein said inhibitor of IL-1β is selected from the group consisting ofan anti-ILβ antibody, a sIL-1R type I, a sIL-1R type II, aninterleukin-1 receptor antagonists and an IL-1 TRAP.
 37. The method ofclaim 20, wherein said inhibitor of IL-1β is an interleukin-1 receptorantagonist.
 38. The method of claim 28, wherein said inhibitor of IL-1βis selected from the group consisting of an anti-ILβ antibody, a sIL-1Rtype I, a sIL-1R type II, an interleukin-1 receptor antagonist, and anIL-1 TRAP.
 39. The method of claim 28, wherein said inhibitor of IL-1βis an interleukin-1 receptor antagonist.